Borrelia burgdorferi, the cauative agent of Lyme disease, is maintained in nature through an infectious cycle between wild mammals and ticks. Like many bacterial pathogens, B. burgdorferi must cope with a changing array of environmental conditions in order to successfully persist, proliferate and be transmitted between hosts. B. burgdorferi has an unusual genomic structure composed of a linear chromosome and a large number of linear and circular plasmids. Abundant evidence indicates that plasmid-encoded genes are critical for adaptation in the infectious cycle. A major focus of our research is to determine the contributions of individual B. burgdorferi genes and plasmids at each stage of the infectious cycle, taking a molecular genetic approach.[unreadable] [unreadable] B. burgdorferi harbors a segmented genome that includes a small 900 kb linear plasmid and as many as 23 circular and linear plasmids, ranging in size from 5kb to 56 kb. The B. burgdorferi genome is unstable during in vitro passage and many of the plasmids can be lost during this process. Loss of certain plasmids is tightly correlated with loss of infectivity and persistence in mice and ticks. For example, we and others have previously demonstrated that the linear plasmid (lp) 25 is critical for infection of the mouse and tick, whereas linear plasmid lp28-1 is required exclusively for persistence in the mouse. Unlike lp25 and lp28-1, the linear plasmid lp36 is not frequently lost by B. burgdorferi during in vitro passage and its potential role in the infectious cycle has not previously been examined. We recently identified a rare B. burgdorferi variant that has lost lp36 but retains all other plasmids known to be important for virulence, thereby allowing investigation of the role of this plasmid in the infectious cycle (Jewett et al. 2007).[unreadable] [unreadable] The lp36 plasmid of the sequenced B. burgdorferi strain B31 is a linear plasmid of approximately 36 kb encoding 54 open reading frames, seven of which appear to be pseudogenes. Most of the genes on lp36 lack homologs in the database and have no predicted function. We found that spirochetes lacking lp36 did not readily survive in the mammal but displayed no deficiency in the tick (Jewett et al. 2007). Mouse infection was restored by reconstitution of the lp36 plasmid in the mutant clone, demonstrating that the infectivity defect resulted from the loss of lp36. Furthermore, we established that the bbk17 gene of lp36 encodes an adenine deaminase (AdeC) and is a genetic component on lp36 that is sufficient to restore mouse infectivity to spirochetes lacking lp36. Our work establishes a critical role for the lp36 plasmid in the infectious cycle (Jewett et al. 2007).[unreadable] [unreadable] The composition of the outer membrane of B. burgdorferi undergoes a profound change as the spirochete is transmitted from the tick vector to a mammalian host. The plasmid-encoded OspA protein is abundant on the surface of B. burgdorferi residing in the midguts of infected ticks but as ticks feed, OspA is down-regulated and replaced with OspC, which is encoded on a different plasmid. This switch in spirochetal surface proteins was hypothesized to be required for migration of B. burgdorferi from the midgut to the salivary glands of the tick or adaptation to the mammalian host after transmission. [unreadable] [unreadable] We previously tested this hypothesis with an investigation of the in vivo role of OspC in both the tick vector and mammalian host. We found that the switch from OspA to OspC is not required for spirochete migration from the midgut to the salivary glands of ticks, but is in preparation for transmission to the mammalian host. Since the essential role of OspC appears to be in establishing mammalian infection, we recently further delineated the critical period in which the protein must be present (Tilly et al. 2007). To address this question, we inoculated wild type and ospC mutant spirochetes into mouse skin and assessed the ability to reisolate spirochetes at various times after inoculation, ranging from 3 hours to 21 days. We also assayed when spirochetes disseminate, by determining when bacteria were first isolated from distal sites. Wild type and complemented mutant spirochetes were isolated from inoculation sites at all time points and first isolated from distant sites at 8 days post-inoculation. In contrast, ospC mutant spirochetes were isolated from inoculation sites through 24 hours post-inoculation (at which time 5/6 mice tested were negative), but never thereafter. We conclude that OspC plays a crucial role in B. burgdorferi infection within the first 48 hours after introduction of spirochetes into the mammal, prior to dissemination or seroconversion of the host (Tilly et al. 2007). [unreadable] [unreadable] The early requirement for OspC excludes a role for the protein in evading the acquired immune reponse and suggests that it is involved in evasion of the host innate immune response. Our working model is that OspC inhibits phagocytosis of B. burgdorferi, perhaps by limiting opsonization by complement, allowing the bacteria to evade clearance immediately after transmission to the mammalian host.[unreadable] [unreadable] [unreadable] These findings, together with previous observations regarding differential regulation of spirochetal gene expression in ticks and mammals, indicate that host adaptation not only occurs in response to disparate arthropod and mammalian environments, but also varies within each host at stages roughly corresponding to colonization, persistence and transmission.